Cytochrome P450 (CYP) enzymes are an important class of heme-containing proteins that catalyze oxidation reactions leading toward the removal of a wide variety of endogenous and exogenous substrates including prescription drugs. The activities of CYP enzymes are regulated primarily at the transcription level involving the regulatory sequences at the 5-flanking region of the CYP genes. The objective of this dissertation study was to characterize the function of the 5-flanking sequences of selected CYP genes primarily responsible for drug metabolism.
Various sequences from the 5-flanking regions of different CYP genes (CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP2E1, and CYP3A4) were cloned in expression vectors and tested for their activity in driving reporter gene expression in mouse livers and in transfected HepG2, 293, and BL-6 cells under optimized conditions. It was demonstrated that among the tested 5-flanking regions of CYP genes, the CYP2D6 promoter showed the highest activity both in vivo and in vitro. The activities of various 5-flanking regions of CYP genes in sustaining transgene expression were then tested in mouse liver and compared to those of other promoter sequences. As a result, the CYP2D6 promoter showed the highest activity and its activity was comparable to that of many established promoters. The mechanism underlying CYP promoter activities in vivo and in vitro were then studied using the CYP2C9 promoter as a model. Activities of various 5-flanking sequences of CYP2C9 were evaluated by using deletion mutations of plasmid constructs in combination with transfection in mouse livers and in HepG2 cells. Finally, the role of PXR and CAR nuclear receptors in regulating CYP2C9 activation was investigated. The results show that both CAR and PXR are essential for CYP2C9 activation and that the regulatory elements reside in the proximal 1-2 kb region upstream of the CYP2C9 gene.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-04072005-160230 |
| Date | 24 May 2005 |
| Creators | Al-Dosari, Mohammed S |
| Contributors | Dr. Joseph E. Knapp, Dr. Paul L. Schiff, Dr. Dexi Liu, Dr. Wen Xie, Dr. Marjorie Romkes |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-04072005-160230/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
Page generated in 0.0026 seconds